Extended Data Fig. 6: cDC1s during homeostasis and tumor challenge in Cd40^cKO and Cd70^cKO mice.

a, Quantification of migratory cDC1 number in TDLNs of tumor-bearing Cd40^WT (Xcr1^+/+ Cd40^fl/fl), Cd40^cKO (Xcr1^Cre/+ Cd40^fl/fl), Cd70^WT (Xcr1^+/+ Cd70^fl/fl), and Cd70^cKO (Xcr1^Cre/+ Cd70^fl/fl) mice as depicted in Fig. 5c. Data represent pooled biologically independent samples from five independent experiments (n = 7-8 for all groups). Data are represented as mean values +/− s.d. **P = 0.0022; ns, not significant. b, Left, Representative flow plots of resident cDC1s (red boxes) and cDC2s (black boxes) in TDLNs of day 6 1956-mOVA-bearing Cd40^WT (Xcr1^+/+ Cd40^fl/fl), Cd40^cKO (Xcr1^Cre/+ Cd40^fl/fl), Cd70^WT (Xcr1^+/+ Cd70^fl/fl), and Cd70^cKO (Xcr1^Cre/+ Cd70^fl/fl) mice. Cells are pregated as B220⁻ CD326⁻MHC-II⁺ CD11c^hi. Numbers are percentages of cells in the indicated gates. Right, quantification of cDC1s as a percentage of resident cDCs. Data represent pooled biologically independent samples from five independent experiments (n = 8 for all groups). Data are represented as mean values +/− s.d. c, Quantification of cDC1s as a percentage of splenic (left), SDLN migratory (middle), and SDLN resident (right) cDCs in WT (Xcr1^+/+ Cd40^fl/fl) and Cd40^cKO mice at homeostasis. Data represent pooled biologically independent samples from five independent experiments (n = 7 for all groups). Data are represented as mean values +/− s.d. ns, not significant. d, Day 6 and Day 14 tumor areas of WT (Xcr1^+/+ Cd70^fl/fl) and Cd70^cKO mice injected with 10⁶ 1956-mOVA cells. Data represent pooled biologically independent samples from three independent experiments (n = 6-8 for WT and n = 10 Cd70^cKO mice). ns, not significant. e, Mitotracker Deep Red FM (right) and Mitotracker Green FM (right) geometric mean MFI in TDLN migratory cDC2s of d6 1956-mOVA-bearing Cd40^WT,Cd40^cKO, Cd70^WT, and Cd70^cKO mice. Data represent pooled biologically independent samples from two independent experiments (n = 4 for all groups). Data are represented as mean values +/− s.d. ns, not significant. f, WT (Xcr1^+/+ Cd40^fl/fl) and Cd40^cKO mice were injected with 10⁶ 1956-mOVA cells. On day 6, mice were injected with the fluorescent activated poly-caspase probe FAM-FLIVO, and then TDLNs were harvested after 1 h. Quantification of FLIVO + cells in migratory and resident cDC1s of WT and Cd40^cKO mice. Data represent pooled biologically independent samples from four independent experiments (n = 7 for WT and n = 8 for Cd40^cKO mice). Data are represented as mean values +/− s.d. *P = 0.0340, ns = not significant. g-h, Basal (g) and maximal (h) OCR from extracellular flux analysis of cDC1s from WT (black), Cd40^-/- (green), and Ptgs2^cKO (red) Flt3L-treated bone marrow cultures. Data represent mean values of four biologically independent experiments. Data are represented as mean values +/− s.d. *P < 0.05. i, Enrichment analysis of mitochondrial complex I biogenesis genes in cDC1 in the absence (red) or presence (blue) of CD40 stimulation. a-b, d-g: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. c: Mann–Whitney test.

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